Studies on a Metal-binding Protein of the Sarcoplasmic Reticulum

Abstract

Abstract The interaction of various cations with the 55,000-dalton protein component of the sarcoplasmic reticulum has been studied by means of equilibrium dialysis and titration with fluorescence and difference absorption spectral techniques. The binding capacities for calcium or strontium are identical, viz. 55 moles per mole of protein, while for zinc the value is 140 moles per mole. It appears that calcium and strontium compete for the same sites, which are also available for zinc. Zinc also binds to additional sites that do not have appreciable affinity for the other cations. Changes in ultraviolet difference absorption spectra and in the intensity of the tryptophan fluorescence produced by binding of calcium, strontium, and zinc in the presence of 0.1 m KCl are correlated with the binding of cations to about 50 sites per molecule; binding of zinc to the additional sites does not entail spectral changes. The ultraviolet difference absorption spectrum developed on binding of divalent cations in the presence of 0.1 m KCl involves mainly tryptophyl residues; KCl alone at concentrations less than 0.1 m produces more complex spectral differences ascribable to several types of chromophores. The changes produced in fluorescence and ultraviolet absorption spectra by KCl at higher concentrations (e.g. 0.6 m) are identical with those obtained by adding divalent cations; no changes are produced by further addition of calcium to 0.6 m KCl. Changes in absorption and fluorescence appear to be due to transfer of aromatic residues from the polar surface to the hydrophobic interior of the protein. This process is, in part, accompanied by an increase in α helix as reflected in measurements of circular dichroism. The order of affinities of various cations on the basis of fluorescence titration is as follows: La3+ g Zn2+ g Cd2+ g Mn2+ g Ca2+ g Mg2+ ≃ Sr2+ >> K+; the affinities range from about 3.3 x 105 for La3+ through 500 for Mg2+ to 4 for K+. The selective removal of protein from the membrane by Triton X-100 is prevented by 4 mm Ca2+. The results have been interpreted in terms of the role the protein may play as a calcium storage site and in reducing the permeability of the sarcoplasmic reticulum.